Adult male C57BL/6 mice (21 g–28 g) were provided by the Animal Center of Chinese Academy of Sciences (Shanghai, China). All procedures were approved by the Institutional Animal Care Committee of the Soochow University and complied with the ARRIVE (Animal Research: Reporting In Vivo Experiments) guidelines. All animals were housed in a quiet and comfortable environment (temperature: 18–22°C, relative humidity: 40%–50%), with a 12 h light/dark cycle. Animals had free access to food and water. Sample numbers were determined by power analysis during the animal ethics dossier application.
Establishment of a controlled cortical impact mouse model
The operation was performed one week after the mice were housed. The TBI model was established by a precision percussion device (68099II, RWD, Shenzhen, China). In brief, mice were anesthetized with isoflurane (3% induction; 1.5% maintenance) and properly fixed in a stereotactic apparatus. A midline incision was made after disinfection to expose the skull. The anterior fontanel was set as the origin, and a coordinate of (2 mm, –2 mm) was set as the center site of craniotomy and impact. An electric drill was used to perform the craniotomy, generating a 3 mm-diameter skull flap, which was removed. During this process, mice with damaged dura were excluded from the experiment. Subsequently, a circular impact tip with a diameter of 2 mm was used to vertically hit the dura mater surface with the following parameters: velocity of 4.5 m/s, depth of 1.0 mm, and duration of 100 ms, resulting in a moderate controlled cortical impact (CCI). Then, the bone flap was returned and the scalp was sutured. The sham operation group received craniotomy, but did not undergo CCI injury. The coronal sections of the brain tissue of the sham group and CCI group are shown in Fig. 1a. The mice were then transferred to the cage and allowed to recover fully from anesthesia (as exhibited by resumption of movement and grooming). During the operation, a 37°C constant heating pad was used to maintain the body temperature of mice.
As mentioned previously, primary cortical neurons (PCNs) from E17 C57BL/6 mouse embryos were isolated and cultured. Briefly, the embryonic mouse brains were removed with sterilized instruments after the pregnant mice were executed. The meninges and blood vessels were removed from the brains of embryonic mice. The bilateral cerebral cortex was taken and the rest of the brain tissue was discarded. The cortical tissue was then digested with 0.25% trypsin-EDTA solution (Gibco, Carlsbad, CA, USA) for 5–8 min at 37°C. After digestion, the tissue was washed three times with phosphate buffered saline (PBS). Fetal bovine serum (FBS; from Gibco) was added to neutralize the trypsin; then, all fluids were filtered and any unfiltered tissue clumps were discarded. The remaining suspension was centrifuged at 1000 rpm/min for 5 min before discarding the supernatant and collecting the lower precipitate. The cell precipitate was resuspended in a tube containing neurobasal medium (from Gibco). The contents of the tube were mixed well to distribute the cells evenly. An appropriate resuspension volume was then drawn on the blood cell count plate and the number of neurons was counted using a microscope. The neurons were then plated onto culture dishes, 6-well plates, or 24-well plates (Corning, NY, USA) precoated with 0.1 mg/ml poly-D-lysine (Sigma-Aldrich, St. Louis, MO, USA) and cultured in fresh neurobasal medium containing 2% B27, 2 mM L-glutamine, 50 U/ml penicillin, and 50 U/ml streptomycin (all from Gibco). The dishes and plates were placed in a 37°C incubator containing 5% CO2. Half of the medium was replaced with fresh medium every 2 days. After transfection and scratching, the neurons were harvested for the following experiments.
Establishment of an in vitro model of scratch injury
As described previously[25, 26], scratch injury, a widely accepted method, was used to establish an in vitro model of TBI. In brief, a sterile pipette tip (10 μl) was used to manually scratch the culture. In 6-well and 24-well plates, 12 × 12 and 6 × 6 scratches were generated, respectively, in each well, and 3 mm × 3 mm grids were formed. The neurons exposed to the tip died immediately, and those far away from the scratches underwent progressive secondary injury. The cells in the control group did not receive this intervention. The injured culture and the corresponding control group were placed in an incubator containing 5% CO2 and humidified air at 37°C for 72 h.
Part 1: Time course analysis of the protein levels of Armcx1 after CCI
In experiment 1, 36 mice (40 in total; 36 survived the surgery) were randomly assigned to six groups with six mice per group. A sham group and five experimental groups were arranged according to the time points of 6 h, 1 d, 3 d, 5 d, and 7 d after CCI. At the specified time point after the surgery, all mice were killed and their brain tissues were collected for the subsequent immunoblotting and immunofluorescence experiments (Fig. 1b).
Part 2: Roles of Armcx1 in secondary brain injury after CCI and the underlying mechanisms in vivo
In this part, the knockdown effect of AAV was verified. Twenty-four mice (27; 3 were excluded) were randomly assigned to the following four groups: pAAV-hSYN-EGFP-miR30shRNA (NC), pAAV-hSYN-EGFP-miR30shRNA(Armcx1)-(1), pAAV-hSYN-EGFP-miR30shRNA(Armcx1)-(2), and pAAV-hSYN-EGFP-miR30shRNA(Armcx1)-(3), with 6 mice per group. Three weeks after injection of the virus, all of the mice in all four groups were sacrificed, and the brain tissues were collected for western blot analysis to identify the virus group with a good knockdown effect for use in the following experiments. Then, 126 mice (136; 10 were excluded) were randomly assigned to the following six groups: sham group, CCI group, CCI + pAAV-hSYN-EGFP group, CCI + pAAV-hSYN-EGFP-Armcx1 group, CCI + pAAV-hSYN-EGFP-miR30shRNA (NC) group, and CCI + pAAV-hSYN-EGFP-miR30shRNA (Armcx1) group. The operation was performed 3 weeks after injection of the virus. The brains of six mice per group were extracted 72 h after CCI for use in western blot, immunofluorescence, and immunohistochemistry. Another three mice in each group were executed 72 h after CCI transmission electron microscopy (TEM), while the remaining 12 mice were examined for behavioral impairment in the week following surgery (Fig. 1c).
Part 3: Further exploration of the mechanism of miR-223-3P/Armcx1 in vitro
As shown in Fig. 1d, the cultured neurons were divided into the following six groups: control group, scratch group, mimic-NC group, mmu-miR-223-3P mimic group, inhibitor-NC group, and mmu-miR-223-3P inhibitor group. Transfection reagents were given 24 h before scratch injury. According to previous time course experiments, the cells were collected at 72 h after scratch injury for western blot, immunofluorescence, and JC-1 staining.
The antibody against Armcx1 (PA5-50911) was purchased from Invitrogen; the Armcx1 antibody (SAB2100153) was from Sigma Aldrich; the antibodies against cleaved caspase-9 (9507), β-actin (4970), and β3-tubulin (4466) were from Cell Signaling Technology; and the anti-NeuN antibody – a neuronal marker (ab104224), NeuN antibody (ab177487), GFAP antibody (ab134436), and Iba1 antibody (ab48004) were from Abcam. The secondary antibodies for western blotting, including goat anti-rabbit IgG-HRP (bs13278) and goat anti-mouse IgG-HRP (bs12478), were purchased from Bioworld. The secondary antibodies for immunofluorescence, including Alexa Fluor 488 donkey anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibody (A21206), Alexa Fluor 555 goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody (A21428), Alexa Fluor 488 goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody (A11001), Alexa Fluor 555 donkey anti-mouse IgG (H+L) highly cross-adsorbed secondary antibody (A31570), Alexa Fluor 555 goat anti-chicken IgY (H+L) secondary antibody (21437), Alexa Fluor Plus 555 donkey anti-goat IgG (H+L) highly cross-adsorbed secondary antibody (A32816), and Alexa Fluor 633 goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody (A-21070) were from Invitrogen.
Injection of the recombinant AAV vector in vivo
Overexpression and knockdown of Armcx1 were achieved by transfection of adeno-associated virus (AAV). To establish and maintain the specific regulation of Armcx1, AAV2/9-hSYN-EGFP-Armcx1 (over-Armcx1) and AAV2/9-hSYN-EGFP-miR30shRNA (Armcx1) (sh-Armcx1) were designed by OBiO (Shanghai, China) and used to up- and down-regulate Armcx1 protein levels, respectively. There were three virus strains in the knockdown group, and the strain with the best knockdown effect was selected for further experiments. Meanwhile, the AAV2/9-hSYN-EGFP (over-NC) and AAV2/9-hSYN-EGFP-miR30shRNA(NC) (sh-NC) were used as the relative negative control. The Armcx1 shRNA sequence was 5′-GGAACAGGACAAGTGGGAA-3′, 5′-CCAACATGACTGTAACTAA-3′, and 5′-GGTGGTCAAAGTGAAAGTT-3′; the shRNA NC sequence was 5’-AGGAAGTCGTGAGAAGTAGAAT-3’. The experimental CCI was established on the 21st day after AAV injection. In brief, mice were anesthetized with isoflurane (3% induction; 1.5% maintenance) and fixed in an appropriate stereotactic frame. A midline scalp incision was made to expose the skull before injecting the virus suspension into the cortex of the mice (0.5 µl over 10 min per site, two sites per mouse). The syringe (Gaoge, Shanghai, China), fitted with a sharp-tip 30-gauge, was placed at the following coordinates: anterior-posterior (AP), bregma 0 mm; medio-lateral (ML), 2.0 mm over the right hemisphere; dorso-ventral (DV), 1.8 mm; bregma, 2.0 mm; medio-lateral (ML), 2.0 mm over the right hemisphere; and dorso-ventral (DV), 1.8 mm from the dura mater. The needle was gently retracted after 10 min to avoid a negative pressure-driven dispersion of the vector solution upward along the needle tract. At the end of the procedure, the scalp was sutured and the mice were allowed to recover on a heating pad maintained at 37°C.
The mmu-miR-223-3P mimic, mmu-miR-223-3P inhibitor was purchased from RiboBio (Guangzhou, China). Transfection of PCNs was performed on the fifth day in 24-well plates in vitro (DIV). Cells were transfected with riboFECTTM CP Reagent (RiboBio, Guangzhou, China) containing the mmu-miR-223-3P mimic, mmu-miR-223-3P inhibitor according to the supplier’s instructions. Twenty-four hours after transfection, the media was replaced with normal conditioned media, and the cells were scratched to establish in vitro model.
In brief, the extracted cells or brain tissue samples around the contusion were collected, homogenized, and mechanically lysed in cold RIPA lysis buffer (Beyotime, Shanghai, China). The samples were then centrifuged for 10 min (4°C, 12000 g). The supernatant was immediately collected and the BCA Protein Assay Kit (Beyotime, Shanghai, China) was used to measure protein concentrations according to the manufacturer’s instructions. Equal amounts of protein samples were loaded onto SDS-polyacrylamide gel, separated, and electrophoretically transferred to a nitrocellulose membrane (Millikon, Spartanburg, SC, USA). Then, the membranes were blocked with 5% bovine serum albumin (BioSharp, Anhui, China) (at room temperature for 1 h), after which the membranes were incubated overnight with primary antibodies at 4°C. GAPDH was also detected and acted as a loading control. The second antibody, coupled with horseradish peroxidase (HRP), was then incubated with TBST for 2 h at room temperature to clean the membranes. The protein bands were displayed by an enhanced chemiluminescence (ECL) Kit (Beyotime, Shanghai, China), and the relative protein quantity was analyzed via ImageJ software (NIH, USA).
For in vivo experiments, the brain samples were fixed in 4% paraformaldehyde, embedded in paraffin, cut into 4 μm sections, and dewaxed immediately before immunofluorescence staining. For in vitro experiments, the cultured neurons were fixed in 4% paraformaldehyde. The sections and cells were then stained with primary antibodies and appropriate secondary antibodies. Nuclei were stained with DAPI mounting medium. Finally, the sections and cells were observed using a fluorescence microscope (Olympus, Tokyo, Japan). At least six random sections of each sample were examined, and the representative results are shown. The relative fluorescence intensity was analyzed using the ImageJ program.
Paraffin-embedded brain sections were dewaxed with gradient ethanol and xylene, and then boiled in a microwave with citrate buffer for 30 min to retrieve antigens. After washing three times with PBS, the sections were incubated with 3% hydrogen peroxide and 3% bovine serum albumin (BSA; BioSharp, Anhui, China) to block endogenous peroxidase and nonspecific binding, respectively. Then, sections were incubated with primary antibodies against β-APP (51-2700, Invitrogen, Carlsbad, CA, USA) overnight at 4°C. The sections were then washed three times with PBS, before incubating with biotinylated secondary antibody in PBS containing 0.3% Triton X-100 for 1 h. Following three washes with PBS, the sections were overlaid with the avidin–biotin horseradish peroxidase (HRP) complex (Vector). Finally, 3,3′-diaminobenzidine solution (DAB; Zsgbbio, Beijing, China) was used to detect the HRP activity under light microscopy. ImageJ software was used to analyze the IHC images.
Transmission electron microscope
A transmission electron microscope (TEM) was used to observe the cortical ultrastructure 3 days after CCI. After the mice were anesthetized and killed, the cortical tissue around the contusion (approximately 1 × 2 mm2) was quickly removed, immediately placed in 2.5% glutaraldehyde, and fixed at 40°C for 4 h. The tissues were rinsed with PBS three times for 10 min each, followed by fixation with 1% osmium acid at 40°C for 2 h. The tissues were rinsed a further three times with PBS buffer. The tissues were then dehydrated in a gradient of 30%, 50%, 70%, 90%, and 100% ethanol for 10 min each, then subject to a further dehydration in 100% ethanol. Subsequently, the samples were embedded with Epon812 epoxy resin and cured at 370°C, 450°C, and 650°C for 24 h each, followed by semi-thin section localization. Ultrathin sectioning was performed using an UltracutE Ultrathin slicer. Tissue was stained with lead uranium-dioxy nitrate acetate. The ultrastructure was observed using a JEM - 1200EX transmission electron microscope (JEOL, Tokyo, Japan).
The mitochondrial membrane potential assay kit with JC-1 (Beyotime, Shanghai, China) was used to detect changes in mitochondrial membrane potential (MMP), which can be used for early detection of cell apoptosis. Tests were performed using the kit according to the manufacturer’s instructions. The culture medium was removed from one well of the 24-well plate, and the cells were washed once with PBS before adding 300 µl cell culture medium. Subsequently, 300 µl JC-1 dyeing solution was added to each well and mixed well before incubating at 37°C for 20 min. Next, an appropriate volume of JC-1 staining buffer (1X) was prepared and placed on ice during incubation. The supernatant was then removed and the cells were washed twice with JC-1 staining buffer (1 X). Subsequently, 500 µl cell culture medium was added. The cells crawled over the slide and were immediately observed under fluorescence microscopy. The red fluorescent complex indicates a higher potential in the mitochondrial membrane. In cells with damaged mitochondria, JC-1 remains in its monomer form and displays green fluorescence. For quantitative analysis, three fields (40×) were randomly selected from three independent trials. The ratio of red to green fluorescence was measured and analyzed by ImageJ.
Modified neurological severity score (mNSS)
The mNSS test was performed to evaluate the neurological functional outcomes of mice. The mNSS test consists of ten tasks that can be used to evaluate the sensory, motor, balance, and reflex functions of mice. Neurological function was graded from 0 to 18, where 0 indicates normal function and 18 indicates maximal deficit. One point was awarded if the mice were unable to perform the test or lacked an expected reaction; thus, the higher the score, the more severe the injury. The mice were trained and assessed before surgery to ensure that the normal score was 0. Then, the tests were conducted blindly and the scores were recorded on days 3, 5, and 7 after CCI.
The rotarod test was used to evaluate motor function after CCI, as described previously. Briefly, mice were placed on an accelerated rotating rod, with the speed increasing linearly from 4 to 40 rpm within 2 min, and maintained for 3 min or until the mice dropped. Each mouse was tested twice a day, with a 15 min interval between tests. The latency to fall off the rotating rod was recorded. Passive rotation, or accompanying the rotating rod without walking, was also considered a fall. Data are expressed as the mean values of the two experiments. The mice were trained three times a day for 3 days before undergoing the operation. The mean value 1 day before the surgery was taken as the baseline. The test was repeated 3, 5, and 7 days after the surgery.
Adhesive removal test
The adhesive removal test was performed to assess the tactile responses and sensorimotor asymmetries of the mice. The sticking plaster (2 × 3 mm) was applied on the left paw (impaired side, contralateral to the brain lesion) as a tactile stimulus. This test was administered on pre-injury days 1, 2, and 3, and post-CCI days 3, 5, and 7. Two trials per day were administered 5 min apart to minimize habituation effects. The baseline latencies to contact and remove the tape were recorded before injury. Tactile responses were measured by recording the time until the initial contact of the impaired forepaw with the mouth, as well as the time to remove the sticking plaster from the impaired forepaw using the mouth, with a maximum observation period of 120 s. The latency to contact and remove the sticking plaster from the left forelimb was the dependent variable of interest.
All data were statistically analyzed using GraphPad Prism 8.0.2 and are presented as the mean ± standard deviation (SD). The data sets in each group were tested for normality of distribution using the Shapiro–Wilk test. The two data groups with normal distributions were compared using the two-tailed unpaired Student’s t-test, and the Mann–Whitney U test was used for the two non-normal data groups. Statistical comparisons between groups were performed using one- or two-way ANOVA and post hoc least significant difference tests for multiple comparisons. P-values < 0.05 were considered statistically significant.